The frequency of occurrence of human fungal infections has been increasing over the past decade in response to a combination of factors (Georgopapadakou et al., 1994). These factors include advances in invasive surgical techniques which allow for opportunistic pathogen access, the administration of immunosuppressive agents employed in transplantation, and an increase in the number of immunosuppressed patients resulting from chemotherapy and disease states such as AIDS. The threat to human health is further compounded by the increased frequency with which resistance to the commonly employed antifungal agents is occurring.
Currently, the most common antifungals include the polyenes and the azoles. The polyenes bind to ergosterol, the fungal membrane sterol, and induce lethal cell leakage (Brajtburg et al., 1990). However, polyenes often have negative side effects and resistance to polyenes has been reported (Hebeka et al., 1965; Powderley et al., 1988). The azoles are fungistatic agents that inhibit the cytochrome P450-mediated removal of the C-14 methyl group from the ergosterol precursor, lanosterol (Vanden Bossche et al., 1987). Resistance to azoles has been reported in Candida albicans (Clark et al., 1996; Sanglard et al., 1996; Sanglard et al., 1995; White, 1997a; White, 1997b) as well as in other species of Candida (Moran et al., 1997; Parkinson et al., 1995), and in other fungal pathogens, including species of Histoplasma (Wheat et al., 1997), Cryptococcus (Lamb et al., 1997; Venkateswarlu al., 1997), and Aspergillus (Denning et al., 1997).
The pathway for fungal sterol biosynthesis is one target for antifungal development. In particular, fungal genes that catalyze a step in sterol biosynthesis that is not found in cholesterol biosynthesis (Pinto et al., 1983) are of interest in this regard. One such fungal gene is the sterol methyltransferase gene (ERG6). Non-recombinant Saccharomyces cerevisiae erg6 mutants have been available for some time (Molzhan et al., 1972). The S. cerevisiae ERG6 gene has been isolated, and recombinant strains prepared (i.e., via genetic engineering) in which the gene has been disrupted (Gaber et al., 1989). Although the absence of the ERG6 gene product in S. cerevisiae was not lethal, it did result in several severely compromised phenotypes (Bard et al., 1978; Kleinhans et al., 1979; Lees et al., 1979; Lees et al., 1980).
S. cerevisiae erg6 mutants have been shown to have diminished growth rates as well as limitations on utilizable energy sources (Lees et al., 1980), reduced mating frequency (Gaber et al., 1989), altered membrane structural features (Kleinhans et al., 1979; Lees et al., 1979), and low transformation rates (Gaber et al., 1989). In addition, several lines of evidence have indicated that S. cerevisiae erg6 mutants have severely altered permeability characteristics. This has been demonstrated using dyes (Bard et al., 1978), cations (Bard et al., 1978), and spin labels used in electron paramagnetic resonance studies (Kleinhans et al., 1979). These early observations have been corroborated recently by the cloning of the S. cerevisiae LIS1 gene (Welihinda et al., 1994), mutants of which were selected on the basis of hypersensitivity to sodium and lithium. Sequencing of LIS1 has indicated identity to ERG6. In addition, studies using the Golgi inhibitor, brefeldin A, have routinely employed erg6 mutants because of their remarkably increased permeability to the compound (Vogel et al., 1993). However, as S. cerevisiae and Candida albicans differ in their ability to survive and grow on various sterol intermediates, and as S. cerevisiae is rarely the cause of a human disease, it was unknown whether the ERG6 gene in the common fungal pathogen, C. albicans, effected similar properties.
Thus, a continuing need exists for fungal genes and strains that can aid in the identification of agents that increase the susceptibility of pathogenic fungi to conventional anti-fungal or anti-metabolic agents.